Purification and properties of glycerol:NAD+ 2-oxidoreductase (glycerol dehydrogenase) from Schizosaccharomyces pombe

Abstract

Glycerol:NAD+ 2-oxidoreductase (glycerol dehydrogenase, EC 1.1.1.6) from Schizosaccharomyces pombe has been purified to homogeneity. The protein has a molecular weight of about 400 000; it can be dissociated into identical subunits of molecular weight 47 000, and is probably an octamer. The pH optimum for glycerol oxidation is 10.0 or higher and for the reverse reaction is 6.0. Oxidation occurs specifically at C2 of glycerol to produce dihydroxyacetone and not glyceraldehyde. Several diols with hydroxyls on adjacent carbon atoms can be oxidized and corresponding carbonyl compounds reduced, 1,2-propanediol being oxidized 1.6 times more rapidly than glycerol. The forward reaction has specific requirement for NAD+ as coenzyme whereas NADPH shows about one-third of the activity of NADH for the reverse reaction. The monovalent cations K+ and NH4+ activate the enzyme, while Na+ and Li+ counteract this effect. Some thiol and chelating agents are inhibitory while thiol antagonists, Mn2+, and to a lesser extent Zn2+, stimulate activity. Apparent K(m) and V(max) values have been determined. The enzyme is similar to but not identical with the glycerol dehydrogenases isolated from Escherichi coli and Klebsiella aerogenes (K. pneumoniae).